Estrogen levels act as a rheostat on p53 levels and modulate p53-dependent responses in breast cancer cell lines Lynnette Fernández-Cuesta, Suresh Anaganti, Pierre Hainaut, Magali Olivier To cite this version: Lynnette Fernández-Cuesta, Suresh Anaganti, Pierre Hainaut, Magali Olivier. Estrogen levels act as a rheostat on p53 levels and modulate p53-dependent responses in breast cancer cell lines. Breast Cancer Research and Treatment, Springer Verlag, 2010, 125 (1), pp.35-42. 10.1007/s10549-010-0819- x. hal-00554995 HAL Id: hal-00554995 https://hal.archives-ouvertes.fr/hal-00554995 Submitted on 12 Jan 2011 HAL is a multi-disciplinary open access L’archive ouverte pluridisciplinaire HAL, est archive for the deposit and dissemination of sci- destinée au dépôt et à la diffusion de documents entific research documents, whether they are pub- scientifiques de niveau recherche, publiés ou non, lished or not. The documents may come from émanant des établissements d’enseignement et de teaching and research institutions in France or recherche français ou étrangers, des laboratoires abroad, or from public or private research centers. publics ou privés. Estrogens levels act as a rheostat on p53 levels and modulate p53–dependent responses in breast cancer cell lines Lynnette Fernández-Cuesta, Suresh Anaganti, Pierre Hainaut, Magali Olivier Molecular Carcinogenesis Group, International Agency for Research on Cancer, Lyon, France Correspondence to: Dr Magali Olivier, Molecular Carcinogenesis Group, International Agency for Research on Cancer, Lyon, France; [email protected]. Running title: Effects of estrogens on p53 levels and activity. Keywords: p53, estrogen receptor, breast, estradiol, doxorubicin, apoptosis 1 Abstract A large proportion of breast cancers express the estrogen receptor alpha (ER ) and are dependent on estrogens for their proliferation and survival. The tumor suppressor TP53 encodes the p53 protein, an important mediator of the anti-proliferative and apoptotic effects of several treatments used for breast cancer. A significant proportions of breast tumors (20-30%) carry mutations in TP53 gene and these mutations are associated with poor survival and poor response to several types of chemotherapeutic treatments. While there is mounting evidence for functional interactions between p53 and ER pathways in breast and other tissues, the impact of these interactions on response to chemotherapy and anti-hormone treat ments remain largely unknown. Here, using estrogen-dependent breast cancer cell lines with different p53 status, we show that estrogens, through ER , influence p53 protein levels and activities. Estrogens deprivation reduced, while estradiol increased p53 levels, in a time and dose-dependent manner. Both wild-type and endogenously expressed mutant p53 proteins were affected. This reduct ion in p53 protein levels resulted in reduced p53-dependent responses induced by DNA-damage in p53 wild-type cells, lowering the capacity of doxorubicine to induce apoptosis. The p53 response appeared to be quantitatively but not qualitatively affected. These results suggest that ER activity is required for a strong p53 response in estrogen-dependent breast cancer cells. These results are in line with previous observations that we made in a clinical series, where a larger effect of TP53 mutation status was found for patient survival in cases with progesterone receptor positive status, a marker of a functional ER pathway. It would thus be important to further characterize the influence of ER pathway on the predictive value of TP53 mutation status in specifically designed clinical trials, as it may open perspectives for improving breast cancer treatment. 2 Introduction The tumour suppressor gene TP53 encodes the p53 protein, a stress induced transcription factor that exerts anti-proliferative activities through the regulation of genes involved in the control of apoptosis, cell cycle checkpoints and senescence [1]. Because TP53 constitutes a major barrier to cancer development it is frequently mutated in various types of human cancers [2]. In breast cancer, TP53 mutations are associated with poor prognosis and several studies have reported an association with poor response to different types of chemotherapy drugs [3]. The estrogen receptor ER is a member of the nuclear receptor family of transcription factors that mediates the effect of estrogens via genomic (i.e. regulation of gene expression requiring specific co-regulator proteins) and non-genomic activities that results in the control of cell growth, survival, differentiation, apoptosis, and angiogenesis [4]. Non-genomic activities include rapid cellular signaling by direct interaction of cytoplasmic ER with a variety of trans- membrane growth factor receptors such as EGFR or HER2 [5;6]. Several lines of evidence suggest that p53 and ER can influence each other activities through different mechanisms. Complexes between p53 and ER proteins have been described that can modulate their stability [7;8]. ER has been shown to control p53 mRNA expression [9;10] and vice-versa [11;12]. p53 can also down-regulate ER - responsive genes by interfering with the binding of ER to its response element (RE) [13- 15], and reciprocally, ER can repress p53 function [16-18]. Positive regulation of ER and p53 may also cooperate in the induction of some genes through p53 and ER RE- half-sites located in promoter regions [19]. Despite these studies, several inconsistent results were reported and the nature, context (ligand-dependent or independent) and biological impacts of these interactions remain to be elucidated. It is well established that p53 participates in the anti-proliferative and apoptotic activities of several anti-cancer drugs that damage DNA, that may account for its prognostic and predictive values in breast cancer [20]. In a large series of 1794 patients with breast cancers we previously observed that the prognostic value of TP53 mutation status was stronger in progesterone receptor (PR) positive cases [21]. Since PR expression is a marker for a functional estrogen receptor pathway, these results suggested that a functional ER pathway may be required for an efficient p53 response in estrogen-dependent breast cancer. To test this hypothesis in cellular models, here we used estrogen-dependent breast cancer cell lines with different TP53 status to investigate how estrogens may influence p53 dependent biological responses. 3 Materials and methods Cell lines and treatments MN1 (p53WT) and MDD2 (p53MUT) cells are isogenic cell-lines derived from MCF-7 cells that have been established in 1992 (kindly provided by M. Oren, Weizmann Institute). MDD2 cells are stably transfected with a p-CMV-DD plasmid expressing a p53 mini protein that contains the first 14 and last 89 amino acids of the mouse p53 protein [22]. This mini p53 protein has strong dominant negative activity due to its hetero- oligomerisation with the endogenous p53 protein. This results in the over expression of an inactive p53 protein in MDD2 cells. MN1 cells were similarly generated by transfection of the control insert-free plasmid followed by G418 selection. MN1 and MCF-7 cells have a similar p53 response when treated with the DNA damaging agent doxorubicin or with estradiol (not shown). These cells were maintained in DMEM (Invitrogen) containing 10%FBS, 2%L-glutamine-penicillin-streptomycin solution stabilised (Sigma) and 0,4mg/ml geneticin (Invitrogen). ZR-75-1 (p53WT) and T-47D (p53MUT, p.L194F) cells were provided by Dr Theillet and BT-474 (p53MUT, p.E285K) by Dr Puisieux. These cells were maintained in RPMI 1640 (Invitrogen). All cells were cultured at 37°C at 5%CO2. For estrogen-free (EF) conditions, cells were cultured in phenol red-free medium (DMEM/F12 –Gibco- and RPMI 1640 –Invitrogen-), supplemented with 10% charcoal- stripped dextran-treated serum (HyClone), and other components as indic ated above. 17β-estradiol (E2) and doxorubicin (DOX) were purchased from Sigma. Cell cycle and apoptosis analyses by Flow Cytometry DNA synthesis was assessed by BrdU (BD-Pharmingen) incorporation and DNA was stained with PI (Sigma) following standard procedures. Cells were fixed with 70% ethanol, DNA denatured with HCl and BrdU revealed with a monoclonal anti-BrdU antibody (BD-Pharmingen). Apoptosis was measured with FITC-Annexin V (BD- Biosciences) following manufacturer’s instructions. Data acquisition and analysis were done on a FACS BDLSRII machine using Cell-Quest software. Each experiment was repeated at least twice. Gene silencing siRNA directed against ER (ID:42835), TP53 (ID:106141), and scramble RNA (scr) which do not recognize any human mRNA (ID:4611) were purchased from Ambion. siRNAs were transfected into cells with HiPerfect (Quiagen) according to the manufacturer’s protocol. ER and p53 mRNA and protein levels were monitored by qPCR and Western-blot respectively. 4 β-galactosidase assay Cells were cultured for 48h in EF medium before transfection, by using FuGene6 reagent (Roche), of p53 reporter plasmid, pRGCΔFosLacZ, containing the RGC (Ribosomal Gene Cluster) p53 binding site upstream of the β-galactosidase. 24h after transfection, different concentrations of E2 were added for 24h and β-galactosidase activity was measured using β-Galactosidase Enzyme Assay (Promega) according to the manufacturer’s instructions. Protein analyses Protein extracts were prepared by lyzing cells with RIPA-like buffer (250mM NaCl, 50mM Tris-HCl pH7.4, 0.1%SDS, 2mM DTT and 0.5% Igepal) containing proteases inhibitors (Complete-Mini, Roche)